Gene therapy vehicles
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This document discusses three physical means of gene transfer: gene guns, electroporation, and microinjection. The gene gun uses compressed gas to propel DNA-coated microprojectiles into target cells, allowing carrier-free delivery. Electroporation uses electric pulses to create temporary pores in the cell membrane to allow DNA entry. Microinjection directly injects DNA solutions into cells through very thin needles under a microscope. While each method has advantages like being targeted or not requiring vectors, they also have limitations such as potential cell damage or requiring specialized equipment and training.
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Gene therapy vehicles
- 2. Biological means of gene transfer Non-biological means of gene transfer Physical means Chemical means
- 3. Physical means of gene transfer Gene gun Electroporation Microinjection
- 4. Gene gun “Particle delivery system, delivers genetic material to target site or cell. It propels nucleic acids on ‘‘bullets’’ to ‘‘shoot’’ genes into cells”
- 5. Gene gun Invented by three scientists: John C Stanford, Ed Wolf, and Nelson Ellen. Also known as particle bombardment, ballistics. originally developed in plant genetics area. Carrier free method. Delivers chemicals, DNA, mRNA, and proteins. BioRad company designs gene gun.
- 6. Components of gene gun He-gas cylinder Firing pin Target cell Gold particles macrocarrier microcarrier vents stopper
- 7. Process of gene transfer by gene gun 1) Preparation of DNA coated beads 2) Precipitation of DNA on microparticles 3) Loading of shells into gun 4) Particle acceleration
- 8. 1) DNA coated beads • Gold particles as beads. Not tungsten. Tungsten is toxic, and not absorbed by cell. • Gold is inert so spermidine is added. • Gold will have a positive charge on it. • Addition of genetic material to gold beads. • Centrifugation at 10,000 r.p.m. • Washing with alcohol. • Addition of PVP.
- 9. 2) Precipitation of DNA on microparticles • Plastic tubes: diameter= 2-4mm length= 2-8 feet • One end of Tube is dipped in PVP tube and other attached with sucker. • Rotation at 180ºC and for 5-8 mins. • Automatic choppers cut tube into 2inch pieces.
- 10. 3) Loading of gun • Short pieces of tubes or shells are loaded in a cartridge of gun. • The tubes have DNA coated beads. • Tubes must have an equal distribution of DNA-beads.
- 11. 4) Particle acceleration • He-gas cylinders provide pressure to gun. • Striker strikes particles which move through the vents and stoppers. • Particles reach the target cell.
- 14. Factors affecting expression rate: 1) Pressure of He-gas cylinders. 195psi for hepatocytes. 200psi for neurocytes. 2) Distance between gun and target cell. distance should be 1inch. 3) Position of gene gun. the angle should be of 90. 4) Ratio of DNA and particles. 5) Ratio of DNA and target cells.
- 15. Gene delivery by naked DNA: Gene delivery by gene gun: Vectored DNA binds cell Enters cytoplasm Enters nucleus Express gene cell binding Enters cytoplasm Enters nucleus Express gene Translocate through cytoplasm Translocate through cytoplasm
- 16. Benefits • Easy to use • Rapid • Absence of toxicity • Targeted delivery • Carrier free Limitations • Best for targets present at surface only. • Can carry limited amount of genetic material ■ Easy to use ■ Rapid ■ Absence of toxicity ■ Targeted delivery ■ Carrier free ■ Easy to use ■ Rapid ■ Absence of toxicity ■ Targeted delivery ■ Carrier free ■ Easy to use ■ Rapid ■ Absence of toxicity ■ Carrier free
- 17. “Electroporation” “A technique in which an electrical field is applied to cells to increase the permeability of the cell membrane.”
- 18. • Physical method of transfection. • allowing chemicals, drugs, or DNA introduction into the cell. • Slow increase in voltage. • 10 times more effective than chemical means. • It mediates transfer of material to multiple cell layers within tissue. • It can transfer more than 50kb fragment.
- 19. • Electroporation is adapted for use in: skeletal muscle. kidney. liver. cardiac tissues. cornea etc. • It gives 100-1000 fold increase in gene expression than micro-injection.
- 21. “Gene delivery by electroporation”
- 22. Plant cell protoplast Foreign DNA Add shock Small Temporary pores created Entry of DNA In cell. Plant cell Cell wall removal
- 24. Benefits • Less costly, fast • Non-toxic • Versatile, Used on all type of cells • Immediate uptake • Not limited by plasmid size • Efficient, easy to use Limitations • Cell damage • Non-specific transport of material • 40-50% of cells are transformed. • Requires optimization for each cell type.
- 25. Applications • Gene/drug delivery to study the effect of gene/drug on cell. • Transgenic mouse development. for research, medical purposes. • Bacterial libraries for protein expression
- 27. “Microinjection” “Microinjection is the direct pressure injection of a solution into cell membrane and nuclear envelope through a glass capillary. It is an effective method.”
- 28. • Well known method, because it is cheap. • Also known as micro-capillaries. • Discovered in early 1990’s. • Dolly sheep. • In-vivo, in vitro, ex vivo targeting. • Composed of syringe having a needle of diameter of 0.5-5µm. • Easy to handle. • Used in: DNA cloning, reproductive cloning,
- 29. • Gene of interest is injected in solution form. • Performed under a specialized optical microscope setup called micro-manipulators.
- 30. Microinjection needle With foreign DNA Holding pipette Nucleus Cytoplasm Needle
- 31. Limitations: • Slow • Expensive • Performed by trained expertise. • More useful for animal cells.
Editor's Notes
- Viral DNA will infect the cell.
- these devices literally propel nucleic acids on ‘‘bullets’’ to ‘‘shoot’’ genes into cells. Beads 0.25-5µm diameter.
- Microcarrier is plate having microparticles. DNA and beads. Macrocarrier takes microcarrier to stoppers with kinetic energy. Velocity depends on mass and velocity.